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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #424531

Research Project: Regulation of Gene Expression in Alfalfa Development and Stress Tolerance

Location: Molecular Plant Pathology Laboratory

2016 Annual Report


Objectives
1. Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic stresses by characterizing gene-stress responses and pathways. Biotic and abiotic stresses cause significant yield losses in alfalfa and greatly reduce the crop’s productivity. Understanding the molecular mechanisms of stress tolerance and the ways by which stress-responsive genes are regulated is essential for improvement of alfalfa adaptability and breeding programs. 2. Aid plant breeders in improving alfalfa productivity and adaptability by implementing genetic and genomic approaches to improve traits related to biotic and abiotic stress tolerance, including, but not limited to, root-knot nematodes and salinity tolerance. Data on stress-responsive genes obtained in this study and other information on alfalfa genomics will be used to identify molecular markers associated with resistance and adaptation to abiotic and biotic stresses in alfalfa.


Approach
The research project will identify stress-responsive gene-candidates in alfalfa and assign them to cognate functional groups related to specific stress responses. It will quantify and confirm roles of the selected genes in adaptation to abiotic and biotic stresses and in regulation of stress responses. Sequence polymorphism in genes underlying stress tolerance will be delineated and molecular markers associated with resistance and adaptation of alfalfa to biotic and abiotic stresses developed. Markers will be validated through cooperative research collaborations.


Progress Report
• Next generation sequencing (NGS) was performed on 36 samples of alfalfa plants susceptible and resistant to bacteria Pseudomonas syringae pv. syringae, a causal agent of bacterial stem blight disease in alfalfa. Expression of alfalfa genes affected by the infection is being analyzed. The goal of the experiment is to investigate molecular mechanisms of alfalfa resistance to bacterial stem blight. • GWAS (genome-wide association study) is underway to develop molecular markers associated with salinity tolerance in alfalfa. • VIGS (virus-induced gene silencing) vector for alfalfa genomic studies is being developed based on the low pathogenic Alfalfa latent carlavirus. Progress is directly related to the Objective 1 of the Project Plan: Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic factors by characterizing gene-stress responses and pathways.


Accomplishments
1. Development of the virus vector for functional genomics studies in alfalfa. The overall goal of this part of the project plan is to develop efficient virus-induced gene silencing (VIGS) vector for alfalfa genomic studies. This novel methodology that is not currently available for alfalfa research will help to gain critical insights for alfalfa breeding programs. The first complete nucleotide sequence of the Alfalfa latent virus (ALV) and the genome structure of the virus were determined and the full-length viral cDNA was cloned into a plasmid vector. RNA transcripts were generated from the full-length viral cDNA which were infectious in the ALV propagation host, Pisum sativum cv. Lincoln. Construction of the infectious full-length cDNA clone of ALV is a necessary step toward development of the ALV-based vector for gene silencing and expression of foreign proteins.

2. Functional characterization of the gene critical for plant development and responses to the environment. Previously, using EST-driven profiling, we identified gene At5G49530 that was associated with defense responses in a model plant Arabidopsis thaliana. Our current research focuses on functional characterization of this gene encoding RPC5 domain of DNA-directed RNA polymerase III and application of this knowledge for alfalfa improvement. To investigate functions of the gene and its possible involvement in adaptive responses to stress, we performed virus-induced silencing (VIGS) of the RPC5 in a model species Nicotiana benthamiana. VIGS led to pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus related to the cellular roles of the subunit. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development.

3. Molecular mechanisms of resistance to bacterial stem blight in alfalfa. Bacterial stem blight of alfalfa caused by Pseudomonas syringae pv. syringae is common in the central and western U.S. and the disease occasionally occurs in eastern states. Yield losses from the disease can be as high as 50% of the first harvest. Our project aims to generate fundamental knowledge on expression and regulation of genes associated with resistance to bacterial stem blight in alfalfa. RNA samples from resistant and susceptible genotypes within two alfalfa accessions were subjected to next generation sequencing and a total of 3,092,825,766 pair-end reads were generated on an Illumina Hi-Seq 2000 platform and assembled into transcripts. Comprehensive gene expression profiling and analysis of transcriptional changes during alfalfa-bacteria interaction provide a dataset of candidate genes involved in resistance to the pathogen. The acquired knowledge will improve our understanding of processes involved in bacterial pathogenicity and host resistance.

4. Genome size dynamics as an important factor for adaptation and evolution of plant species. Genome size of the eukaryotic organisms is a fundamental characteristic that critically affects adaptation of the species and plays an important role in plant phenotypic evolution. In collaboration with Dr. A.M. Boutanaev of the Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, we investigated genome size dynamics in multiple genera of the diploid seed plants and found a remarkably strong correlation between genome size of the species and their evolutionary age. This linearity of the intrageneric genome size dynamics may have persisted over millions of years probably due to the gradual changes in DNA content unrelated to natural selection process. The findings will contribute to the basic understanding of mechanisms involved in the modern-day adaptive evolution in plants.


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Review Publications
Postnikova, O., Shao, J.Y., Mock, N.M., Baker, C.J., Nemchinov, L.G. 2015. Gene expression profiling in viable but not culturable (VBNC) cells of Pseudomonas syringae pv syringae. Frontiers in Microbiology. 6:1419.
Nemchinov, L.G., Boutanaev, A.M., Postnikova, O.A. 2016. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana. Scientific Reports. 6:27785.